Methods for asymmetric semi-nested isothermal nucleotide amplification

This application is a non-provisional and claims benefit of U.S. Provisional Application No. 63/171,761 filed Apr. 7, 2021 and U.S. Provisional Application No. 63/240,227 filed Sep. 2, 2021, the specification(s) of which is/are incorporated herein in their entirety by reference.

This application is a non-provisional and claims benefit of U.S. Provisional Application No. 63/183,504 filed May 3, 2021, the specification of which is incorporated herein in their entirety by reference.

The present invention features an asymmetric semi-nested isothermal nucleotide amplification (ANINA) method, as well as compositions and systems, for the amplification of single-stranded oligonucleotides from a target sequence.

Nucleic acid testing (NAT) biosensing technologies, such as those employing the use of fluorophores or redox sensors or other physicochemical detectors, are currently used in numerous applications for the detection of nucleic acids (NA) of a particular pathogen or mutated genes in an organism. NATs use the specificity and sensitivity afforded by nucleic acid base pairing to detect different NA sequences, sometimes differing by a single nucleotide. However, for a NAT to work effectively, single-stranded DNA and/or RNA need to be isolated for detection. This allows for a physicochemical detector (e.g., a genosensor probe) to hybridize to its complementary sequence, which should be a single-stranded DNA and/or RNA target.

This can be achieved by fragmenting the genomic DNA/RNA or by amplifying the target sequence using nucleic acid amplification technologies (NAATs), which increases the number of target copies and the sensitivity of the detection. However, both of the aforementioned methods have shortcomings associated with them.

First, fragmenting DNA in a controlled manner to a size between 25-100 bps is extremely difficult to achieve. Most mechanical and chemical breakdown methods give ˜1 kbps fragments which are not preferred as longer sequences are more difficult to denature and tend to form secondary structures, thereby decreasing the hybridization efficiency. Additionally, enzymatic methods usually use DNAses that have a propensity of depolymerizing DNA to fragments less than 25 bps. Even the commercially available DNAses, such as fragmentase (NEB), are difficult to control to give size-specific fragments. Lastly, the nucleic acid fragments need to be denatured, if they are double-stranded, to bind to the single-stranded probe, leading to competition between the probe and homologous strand which makes the process more complicated and inefficient. Furthermore, there is the potential for non-specific binding to the probe of other fragments.

The most used NAAT is PCR which remains the gold standard of current diagnostics. However, PCR is difficult to conduct in a point-of-need setting, because of the requirement of specific cycling temperatures: for example, (i) a very high temperature (usually 95° C.) required for thermal melting of dsDNA/dsRNA (either the starting sequence or the amplicons after the first amplification cycle), (ii) annealing temperature (usually between 50-70° C.) for the primers to bind to, and (iii) the extension temperature for the corresponding amplification by the common polymerases (e.g., Taq (72° C.)). Additionally, PCR and real-time PCR are limited to the time required for each cycle, thereby increasing the total time of the reactions. Other isothermal NAATs, such as LAMP (loop-mediated isothermal amplification), HDA (helicase-dependent isothermal DNA amplification). SDA (strand displacement amplification), NASBA (nucleic acid sequence-based amplification), RCA (rolling circle amplification), can amplify DNA in a simpler setting by avoiding the need for a cycling method. However, all the aforementioned methods still require either thermal melting of the dsDNA or dsRNA, or incubation at a temperature higher than 50° C. or require the addition of finicky nucleases adding to the complexity of the process.

Additionally, most NAATs can amplify if there is a contamination, and therefore nested amplification strategies are utilized to make the amplicon results more specific. Furthermore, most NAATs utilize a dual strand amplification strategy to increase the number of copies exponentially. However, this results in dsDNA amplicons, which still need to be denatured before being detected. While asymmetric amplification strategies can be used to amplify mostly single-stranded DNA, it heavily limits the amplification rate to a linear rate instead of the exponential rate of the dual-strand approach.

Currently, an isothermal cost-effective nucleic acid amplification technology (NAAT) that specifically amplifies single-strand oligonucleotides is needed. Furthermore, a method that does not require thermal melting and can be done in a point-of-need setting would be ideal.

It is an objective of the present invention to provide systems, compositions, and methods (e.g., asymmetric semi-nested isothermal nucleotide amplification (ANINA)) that allow for amplifying single-stranded oligonucleotides. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

The present invention features a method of real time asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing and quantifying single-stranded oligonucleotide amplicons. The method comprises introducing to a sample a set of primers, e.g., three primers. e.g., a first primer, a second primer, and a third primer.,, andoutlines the origin of the sequences of the primers and their relationship to the target region. In some embodiments, the first primer refers to a first forward primer (FP1), the second primer refers to a second forward primer (FP2), and the third primer refers to a reverse primer (RP). In some embodiments, the first primer refers to a first reverse primer (RP1), the second primer refers to the second reverse primer (RP2), and the third primer refers to the forward primer (FP). Thus, as used herein, the first forward primer is also known as the first primer, and the second forward primer is also known as the second primer, and the reverse primer is also known as the third primer. As used herein, the first reverse primer is also known as the first primer, the second reverse primer is also known as the second primer, and the forward primer is also known as the third primer.

In some embodiments, the ratio of the first primer (P1):second primer (P2):third primer (P3) (e.g., FP1:FP2:RP or RP1:RP2:FP) is (1-10):(10-200):(1-10). In some embodiments, the ratio of P1:P2:P3 is 1:(50-100):3.

In some embodiments, the method comprises introducing to a sample a solution comprising one or more enzymes, dNTPs, one or more buffering reagents, one or more salts, and one or more crowding reagents and a reporter probe. In some embodiments, the method comprises incubating the sample with the primers and solution at a reaction temperature for a length of time. In some embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the RP and a sequence extending from and including at least a portion of the FP2. In other embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the FP and a sequence extending from and including at least a portion of the RP2. In some embodiments, the method quantifies the single-stranded oligonucleotide amplicons produced when the single-stranded oligonucleotide amplicons bind to the reported probe.

The present invention features a method of asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing single-stranded oligonucleotide amplicons. In some embodiments, said method comprises introducing to a sample 1) a set of primers comprising a first forward primer (FP1), a second forward primer (FP2), and a reverse primer (RP). In some embodiments. FP2 is downstream of FP1 In other embodiments, said method comprises introducing to a sample 1) a set of primers comprising a first reverse primer (RP1), a second reverse primer (RP2), and a forward primer (FP). In some embodiments, RP2 is upstream of RP1. In some embodiments, the ratio of P1:P2:P3 (e.g., FP1:FP2:RP and/or RP1:RP2:FP) is 1:(50-100):3. In some embodiments, the ratio of P1:P2:P3 (e.g., FP1:FP2:RP and/or RP1:RP2:FP) is (1-10):(10-200):(1-10). In some embodiments, the method comprises introducing to a sample 2) a solution comprising enzymes, dNTPs. and a buffer comprising a buffering agent, salts, and crowding reagents. In some embodiments, the method comprises incubating the sample with the primers and the solution at a reaction temperature for a length of time. In some embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the RP and a sequence extending from and including at least a portion of the FP2. In other embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the FP and a sequence extending from and including at least a portion of the RP2.

In some embodiments, the present invention features a method of point-of-care amplification of a target sequence. In some embodiments, said method comprises introducing to a sample 1) a set of primers comprising a first forward primer (FP1), a second forward primer (FP2), and a reverse primer (RP). In some embodiments, FP2 is downstream of FP1. In other embodiments, said method comprises introducing to a sample 1) a set of primers comprising a first reverse primer (RP1), a second reverse primer (RP2), and a forward primer (FP). In some embodiments, RP2 is upstream of RP1. In some embodiments, the ratio of P1:P2:P3 (e.g., FP1:FP2:RP and/or RP1:RP2:FP) is 1:(50-100):3. In other embodiments, the ratio of P1:P2:P3 (e.g., FP1:FP2:RP and/or RP1:RP2:FP) is (1-10):(10-200):(1-10). In some embodiments, said method comprises introducing to a sample 2) a solution comprising enzymes, dNTPs. and a buffer comprising a buffering agent, salts, and crowding reagents. In some embodiments, the method comprises incubating the sample with the primers and solution at a reaction temperature for a length of time. In some embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the RP and a sequence extending from and including at least a portion of the FP2. In other embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the FP and a sequence extending from and including at least a portion of the RP2.

In some embodiments, the present invention features a kit for amplifying single-stranded oligonucleotides. In some embodiments, said kit comprises: a recombinase, a single-stranded binding protein, strand displacing polymerase, dNTPs, and a buffer. In some embodiments, the buffer comprises buffering agents, salts, and crowding reagents. In some embodiments, said kit comprises: a first primer (P1), a second primer (P2), and a third primer (P3), e.g., a first forward primer (FP1), a second forward primer (FP2), wherein FP2 is downstream from FP1, and a reverse primer (RP) or a first reverse primer (RP1), a second reverse primer (FP2), wherein RP2 is upstream from RP1, and a forward primer (FP). In some embodiments, the ratio of P1:P2:P3 (e.g., FP1:FP2:RP and/or RP1:RP2:FP) is 1:(50-100):3. In other embodiments, the ratio of P1:P2:P3 (e.g., FP1:FP2:RP and/or RP1:RP2:FP) is (1-10):(10-200):(1-10). In some embodiments, the kit further comprises a reverse transcriptase enzyme.

The present invention features a system for performing asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing single-stranded oligonucleotide amplicons as described herein. In some embodiments, said system comprises: a kit for amplifying single-stranded oligonucleotides as described herein and a reaction chamber for accepting the kit and a sample. In some embodiments, the reaction chamber is configured to incubate the kit and sample at a reaction temperature for a length of time such that the asymmetric semi-nested isothermal nucleotide amplification (ANINA) system amplifies a single-stranded amplicon therein.

One of the unique and inventive technical features of the present invention is the use of a first forward primer (FP1), a second forward primer (FP2), wherein FP2 is downstream from FP1, and a reverse primer (RP) or the use of a first reverse primer (RP1), a second reverse primer (RP2) wherein RP2 is upstream from RP1, and a forward primer (FP1) in addition to the use of a specific ratio of the forward primers to reverse primer for each primer set. Additionally, the primer design process is similar to that of PCR, instead of complicated methods used for other INAs, such as LAMP, RCA, SIBA. The semi-nested approach also increases the specificity of the amplification over a non-nested approach, while the specific ratio of the primers increases the amplification rate of the single strand target region compared to traditional asymmetric amplification strategies. Additionally. In this system there is no need for ATP or ATP-γ-S or an additional ATP regeneration system for the ATPases such as recombinase, such as those required in recombinase polymerase amplification (RPA) or strand invasion based amplification (SIBA). Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for a method of producing mostly single-stranded oligonucleotide amplicons. None of the presently known prior references or work has the unique inventive technical feature of the present invention.

Furthermore, the inventive technical features of the present invention contributed to a surprising result. For example, the present invention features an amplification system that did not need a separate addition of ATP or ATP-γ-S. While additional dATP helped with the amplification, amplification without the need of addition of any dATP, apart from the dATP already present in the dNTP mix was also observed. Additionally, the methods and systems described herein can be combined with a NAT platform which uses a sensitive detection technology, and can detect the amplicons in real time. This can translate to give information about not only the presence of a specific target gene or gene polymorphisms, but the amount of the said target as well.

In some embodiments, this method can also be combined with any other system that requires a specific amplification of a single-stranded oligonucleotide target for detection or a part of another experiment, such as gene silencing. In other embodiments, this method can be expanded for multiplex amplification of multiple target sequences.

As previously discussed, the present invention features methods of asymmetric semi-nested isothermal nucleotide amplification for producing single-stranded oligonucleotide amplicons of a target region of nucleic acid. In some embodiments, the method comprises introducing to a sample a set of primers and a solution comprising enzymes, dNTPs, and a buffer comprising buffering reagents, salts, and crowding reagents, and incubating the sample with the primers and solution at a reaction temperature for a length of time. The set of primers may comprise a first primer (P1), P1 has a sequence that is a set of nucleotides 5′ to the target region, wherein P1 binds to a first complementary binding region (CSBR1) which is on a strand opposite the target region; a second primer (P2), P2 has a sequence that is a set of nucleotides (a) 5′ to the target region or (b) 5′ to the target region and including a portion of the target region, wherein P2 binds to a second complementary binding region (CSBR2) which is on a strand opposite the target region; wherein the set of nucleotides for P1 is at least partially 5′ to the set of nucleotides for P2; and a third primer (P3), P3 has a sequence that is a set of nucleotides complementary to (a) at least a portion of the target region, or (b) a portion of the 3′ end of the target region and one or more nucleotides downstream of the 3′ end of the target region; or (c) an area 3′ to the target region; wherein P3 binds to a target strand binding region (TSBR) of the target strand. The ratio of P1:P2:P3 may be (1-10):(20-200):(1-20). The method produces single-stranded oligonucleotide amplicons having a sequence comprising at least the target region.

In some embodiments, the ratio of P1:P2:P3 is 1:(50-100):3. In some embodiments, the target region is from 20 to 500 bases in length. In some embodiments, the buffer further comprises a reducing agent. In some embodiments, the enzymes comprise a recombinase enzyme, a single strand binding protein, a strand displacing polymerase, a reverse transcriptase or a combination thereof. In some embodiments, the recombinase is RecA, or Rad51, or RadA. In some embodiments, the single-stranded binding protein iscol single-stranded DNA binding protein (EcSSB). In some embodiments, the strand displacing polymerase isDNA polymerase I (Bsu), or mesophilic DNA polymerase. In some embodiments, the buffering reagents are Tris, PBS, or a combination thereof. In some embodiments, the salts comprise sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl), sodium acetate (NaCHCOO), magnesium acetate (Mg(CHO), monosodium phosphate (NaH2PO4), disodium phosphate (NAPO), or a combination thereof. In some embodiments, the crowding agent is polyvinylpyrrolidone (PVP), or polyethylene glycol (PEG), Ficoll, Dextran, or a combination thereof. In some embodiments, the reaction temperature ranges from 15° C. to 60° C. In some embodiments, the length of time is from 5 to 60 minutes. In some embodiments, the buffer has a pH ranging from 7.0-8.0. In some embodiments, the method further comprises detecting the target sequence. In some embodiments, detection of the target sequence features introducing a genosensor probe.

The present invention also features methods of real time asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing and quantifying single-stranded oligonucleotide amplicons of a target region of nucleic acid. In some embodiments, the method comprises introducing to a sample a set of primers, a solution comprising enzymes, dNTPs, and a buffer comprising buffering reagents, salts, and crowding reagents; and a reporter probe; and incubating the sample with the primers and solution at a reaction temperature for a length of time. In some embodiments, the set of primers comprises a first primer (P1), P1 has a sequence that is a set of nucleotides 5′ to the target region, wherein P1 binds to a first complementary binding region (CSBR1) which is on a strand opposite the target region; a second primer (P2), P2 has a sequence that is a set of nucleotides (a) 5′ to the target region or (b) 5′ to the target region and including a portion of the target region, wherein P2 binds to a second complementary binding region (CSBR2) which is on a strand opposite the target region; wherein the set of nucleotides for P1 is at least partially 5′ to the set of nucleotides for P2; and a third primer (P3), P3 has a sequence that is a set of nucleotides complementary to (a) at least a portion of the target region, or (b) a portion of the 3′ end of the target region and one or more nucleotides downstream of the 3′ end of the target region; or (c) an area 3′ to the target region; wherein P3 binds to a target strand binding region (TSBR) of the target strand. In some embodiments, the ratio of P1:P2:P3 is (1-10):(20-200):(1-20). The method produces single-stranded oligonucleotide amplicons having a sequence comprising at least the target region. The method may quantify the single-stranded oligonucleotide amplicons produced when the single-stranded oligonucleotide amplicons bind to the reported probe.

In some embodiments, the one or more enzymes comprises a recombinase enzyme, a strand displacing polymerase, a reverse transcriptase, or a combination thereof, in some embodiments, the recombinase enzyme is RecA. In some embodiments, the strand displacing polymerase isDNA polymerase I (Bsu), Bst, or Klenow Fragment. In some embodiments, the buffering reagents are Tris, PBS, or a combination thereof. In some embodiments, the one or more salts is magnesium acetate (Mg(CHO)or (Mg(CHO)and one or a combination of: sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl), sodium acetate (NaCHCOO), monosodium phosphate (NaHPO), and disodium phosphate (NaPO). In some embodiments, the one or more crowding reagents comprises polyvinylpyrrolidone (PVP) or PVP and one or a combination of: polyethylene glycol (PEG), Ficoll, and Dextran. In some embodiments, the solution further comprises a single stranded binding protein (SSB). In some embodiments, the SSB is T4 gp32 SSB,SSB (EcSSB), orDNA polymerase I (Bsu). In some embodiments, the solution further comprises a reducing agent. In some embodiments, the sample is in a buffer comprising 20 mM PBS, 2.5 mM EDTA, and 0.05% SDS.

Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

Before the present compounds, compositions, and/or methods are disclosed and described, it is to be understood that this invention Is not limited to specific synthetic methods or to specific compositions, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

For purposes of summarizing the disclosure, certain aspects, advantages, and novel features of the disclosure are described herein. It is to be understood that not necessarily all such advantages may be achieved in accordance with any particular embodiments of the disclosure. Thus, the disclosure may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

Additionally, although embodiments of the disclosure have been described in detail, certain variations and modifications will be apparent to those skilled in the art, including embodiments that do not provide all the features and benefits described herein. It will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative or additional embodiments and/or uses and obvious modifications and equivalents thereof. Moreover, while a number of variations have been shown and described in varying detail, other modifications, which are within the scope of the present disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the present disclosure. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the present disclosure. Thus, it is intended that the scope of the present disclosure herein disclosed should not be limited by the particular disclosed embodiments described herein.

Referring now to the figures, the present invention features systems, compositions, and methods (e.g., real-time asymmetric semi-nested isothermal nucleotide amplification (ANINA)) that allows for amplifying single-stranded oligonucleotides.

,, andoutlines the origin of the sequences of the primers and their relationship to the target region. In some embodiments, the first primer refers to a first forward primer (FP1), the second primer refers to a second forward primer (FP2), and the third primer refers to a reverse primer (RP). In some embodiments, the first primer refers to a first reverse primer (RP1), the second primer refers to the second reverse primer (RP2), and the third primer refers to the forward primer (FP). Thus, as used herein, the first forward primer is also known as the first primer, and the second forward primer is also known as the second primer, and the reverse primer is also known as the third primer. As used herein, the first reverse primer is also known as the first primer, the second reverse primer is also known as the second primer, and the forward primer is also known as the third primer.

In some embodiments, the first primer (P1) and second primer (P2) share similar nucleotides e.g., the 3′ end (e.g., the last nucleotide, the last two, the last three, last four, last five, etc.) of P1 is the same as the 5′ end (the first nucleotide, the first two, the first three, first four, first five, etc.) of P2. In some embodiments, the first primer (P1) and second primer (P2) do not overlap. In some embodiments, the first primer (P1) and second primer (P2) are spaced a distance apart. e.g., the sequences are from two regions of the strand that are at least 1 nucleotide apart, e.g., 1 nucleotide apart, 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 25, 30, 30-40, 40-50, 50-75, 75-80, 80-100, 100-150, 150-200, more than 200, etc.

In some embodiments, the second primer (P2) and the target region share similar nucleotides e.g., the 3′ end (e.g., the last nucleotide, the last two, the last three, etc.) of P2 is the same as the 5′ end (the first nucleotide, the first two, the first three, etc.) of the target region. In some embodiments, the second primer (P2) and the target region do not overlap. In some embodiments, the second primer (P2) and the target region are spaced a distance apart. e.g., the sequences are from two regions of the strand that are at least 1 nucleotide apart, e.g., 1 nucleotide apart, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 17, 18, 19 20, 21, 22, 23, 24, 25, 25-30, 30-40, 40-50, 50.75, 75-80, 80-100, 100-150, 150-200, more than 200, etc.

In some embodiments, the third primer (P2) and the nucleotides complementary to the target region share similar nucleotides e.g., the 3′ end (e.g., the last nucleotide, the last two, the last three, etc.) of P3 is the same as the 5′ end (the first nucleotide, the first two, the first three, etc.) of the nucleotides complementary to the target region. In some embodiments, the third primer (P3) and the nucleotides complementary to the target region do not overlap. In some embodiments, the third primer (P3) and the nucleotides complementary to the target region are spaced a distance apart, e.g., the sequences are from two regions of the strand that are at least 1 nucleotide apart, e.g., 1 nucleotide apart, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 25-30, 30-40, 40-50, 50-75, 75-80, 80-100, 100-150, 150-200, more than 200, etc.

In some embodiments, the target region is from 20 to 500 bases in length. In other embodiments, the target region is from 20 to 400 bases in length. In some embodiments, the target region is from 20 to 300 bases in length. In some embodiments, the target region is from 20 to 200 bases in length. In some embodiments, the target region is from 20 to 100 bases in length. In some embodiments, the target region is from 20 to 50 bases in length. In some embodiments, the target region is from 20 to 25 bases in length. In some embodiments, the target region is from 25 to 500 bases in length. In some embodiments, the target region is from 25 to 400 bases in length. In some embodiments, the target region is from 25 to 300 bases in length. In some embodiments, the target region is from 25 to 200 bases in length. In some embodiments, the target region is from 25 to 100 bases in length. In some embodiments, the target region is from 25 to 50 bases in length. In some embodiments, the target region is from 50 to 500 bases in length. In some embodiments, the target region is from 50 to 400 bases in length. In some embodiments, the target region is from 50 to 300 bases in length. In some embodiments, the target region is from 50 to 200 bases in length. In some embodiments, the target region is from 50 to 100 bases in length.

outlines the strategy of the methods hereon. For example, as shown on the left side of, the first primer (P1) and third primer (P3) bind to the complementary strand and target strand, respectively, the first primer (P1) binds to the first complementary strand binding region (CSBR1) and the third primer (P3) binds to the target strand binding region (TSBR). As shown on the right side of, the second primer (P2) and third primer (P3) bind to the complementary strand and target strand, respectively; the second primer (P2) binds to the second complementary strand binding region (CSBR2) and the third primer (P3) binds to the target strand binding region (TSBR). The products are shown below. As the reactions proceed, the longest product of dsDNA is produced in the least amount (left side of), and the shorter products are produced in greater amounts relative to the longest product; the highest yield is the single stranded oligonucleotide amplicon generated by P2 and P3 (see bottom of). This contains the target region and includes the target strand binding region. Note the sizes listed inare examples only. The present invention is not limited to an 88 base amplicon.

In some embodiments, the single-stranded oligonucleotide amplicons are from 20 to 500 bases in length. In other embodiments, the single-stranded oligonucleotide amplicons are from 20 to 400 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 20 to 300 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 20 to 200 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 20 to 100 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 20 to 50 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 20 to 25 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 25 to 500 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 25 to 400 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 25 to 300 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 25 to 200 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 25 to 100 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 25 to 50 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 50 to 500 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 50 to 400 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 50 to 300 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 50 to 200 bases in length. In some embodiments, the single-stranded oligonucleotide amplicons are from 50 to 100 bases in length.

Primer lengths are well known to be an ordinary skill in the art. For example, in some embodiments, P1 or P2 or P3 is from 18 to 30 bases in length, e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 bases in length. The present invention is not limited to these primer lengths. In some embodiments, the primers may be less than 18 bases in length. In some embodiments the primers may be greater than 30 bases in length. For example, the primers may be from 30-40 bases in length, or more. Primers may be longer for various reasons such as but not limited to the user of an adapter.

In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is from 18 to 25 bp upstream of the first nucleotide (5′ end) of the target region. In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is from 20 to 30 bp upstream of the first nucleotide (5′ end) of the target region. In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is from 30 to 40 bp upstream of the first nucleotide (5′ end) of the target region. In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is from 40 to 50 bp upstream of the first nucleotide (5′ end) of the target region. In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is from 50 to 60 bp upstream of the first nucleotide (5′ end) of the target region. In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is from 60 to 70 bp upstream of the first nucleotide (5′ end) of the target region. In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is from 70 to 80 bp upstream of the first nucleotide (5′ end) of the target region. In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is from 80 to 100 bp upstream of the first nucleotide (5′ end) of the target region.

In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is from 100 to 200 bp upstream of the first nucleotide (5′ end) of the target region. In some embodiments, the sequence of the first nucleotide of P1 (e.g., the 5′ end of P1) is more than 200 bp upstream of the first nucleotide (5′ end) of the target region. The present invention is not limited to the aforementioned positional relationships.

In some embodiments, the sequence of the first nucleotide of P3 (e.g., the 5′ end of P3) is from 0 to 5 bp upstream of the first nucleotide (5′ end) of the complement of the target region. In some embodiments, the sequence of the first nucleotide of P3 (e.g., the 5′ end of P3) is from 5 to 15 bp upstream of the first nucleotide (5′ end) of the complement of the target region. In some embodiments, the sequence of the first nucleotide of P3 (e.g., the 5′ end of P3) is from 15 to 25 bp upstream of the first nucleotide (5′ end) of the complement of the target region. In some embodiments, the sequence of the first nucleotide of P3 (e.g., the 5′ end of P3) is from 25 to 50 bp upstream of the first nucleotide (5′ end) of the complement of the target region. In some embodiments, the sequence of the first nucleotide of P3 (e.g., the 5′ end of P3) is from 50 to 75 bp upstream of the first nucleotide (5′ end) of the complement of the target region. In some embodiments, the sequence of the first nucleotide of P3 (e.g., the 5′ end of P3) is from 75 to 100 bp upstream of the first nucleotide (5′ end) of the complement of the target region. In some embodiments, the sequence of the first nucleotide of P3 (e.g., the 5′ end of P3) is more than 100 bp upstream of the first nucleotide (5′ end) of the complement of the target region. The present invention is not limited to the aforementioned positional relationships.

The present invention features a method of real time asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing and quantifying single-stranded oligonucleotide amplicons. In some embodiments, the method comprises introducing to a sample a set of primers. In some embodiments, the set of primers comprise a first forward primer (FP1), a second forward primer (FP2), and a reverse primer (RP). In some embodiments, the FP2 is downstream of FP1. In some embodiments, the ratio of P1:P2:P3 (e.g., FP1:FP2:RP) is (1-10):(10-200):(1-10). In other embodiments, the ratio of P1:P2:P3 (e.g., FP1:FP2:RP) is 1:(20-200):(2-10). In further embodiments, the ratio of FP1:FP2:RP is 1:(50-100):3. In some embodiments, the method comprises introducing to a sample a solution comprising one or more enzymes, dNTPs, one or more buffering reagents, one or more salts, and one or more crowding reagents and a reporter probe. In some embodiments, the method comprises incubating the sample with the primers and solution at a reaction temperature for a length of time. In some embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the RP and a sequence extending from and including at least a portion of the FP2. In some embodiments, the method quantifies the single-stranded oligonucleotide amplicons produced when the single-stranded oligonucleotide amplicons bind to the reported probe.

The present invention may also feature a method of real time asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing and quantifying single-stranded oligonucleotide amplicons. In some embodiments, the method comprises introducing to a sample a set of primers. In some embodiments, the set of primers comprise a reverse primer (RP1), a second reverse primer (RP2) and a forward primer (FP). In some embodiments, the RP2 is upstream of RP1. In some embodiments, the ratio of RP1:RP2:FP is (1-10):(10-200):(1-10). In other embodiments, the ratio of RP1:RP2:FP1 is 1:(20-200):(2-10). In further embodiments, the ratio of RP1:RP2:FP is 1:(50-100):3. In some embodiments, the method comprises introducing to a sample a solution comprising one or more enzymes, dNTPs, one or more buffering reagents, one or more salts, and one or more crowding reagents and a reporter probe. In some embodiments, the method comprises incubating the sample with the primers and solution at a reaction temperature for a length of time. In some embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the FP and a sequence extending from and including at least a portion of the RP2. In some embodiments, the method quantifies the single-stranded oligonucleotide amplicons produced when the single-stranded oligonucleotide amplicons bind to the reported probe.

In some embodiments, the reporter probe is a fluorescent probe. In other embodiments, the reporter probe is a redox probe.

The present invention features a method of asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing single-stranded oligonucleotide amplicons. In some embodiments, said method comprises introducing to a sample 1) a set of primers comprising a first forward primer (FP1), a second forward primer (FP2), and a reverse primer (RP). In some embodiments. FP2 is downstream of FP1 In some embodiments, the ratio of FP1:FP2:RP is (1-10):(10-200):(1-10). In other embodiments, the ratio of FP1:FP2:RP is 1:(20-200):(2-10). In further embodiments, the ratio of FP1:FP2:RP is 1:(50-100):3. In some embodiments, the method comprises introducing to a sample 2) a solution comprising enzymes. ATP, dNTPs, and a buffer comprising buffering agents, salts, crowding reagents, and reducing reagents. In other embodiments, the method comprises introducing to a sample 2) a solution comprising enzymes, dATP, dNTPs, and a buffering agent comprising salts, crowding reagents, and reducing reagents. In some embodiments, the method comprises incubating the sample with the primers and solution at a reaction temperature for a length of time. In some embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the RP and a sequence extending from and including at least a portion of the FP2.

The present invention features a method of asymmetric semi-nested isothermal nucleotide amplification (ANINA) for producing single-stranded oligonucleotide amplicons. In some embodiments, said method comprises introducing to a sample 1) a set of primers comprising a first reverse primer (RP1), a second reverse primer (RP2), and a forward primer (FP). In some embodiments, RP2 is upstream of RP1. In some embodiments, the ratio of RP1:RP2:FP is (1-10):(10-200):(1-10). In other embodiments, the ratio of RP1:RP2:FP1 is 1:(20-200):(2-10). In further embodiments, the ratio of RP1:RP2:FP is 1:(50-100):3. In some embodiments, the method comprises introducing to a sample 2) a solution comprising enzymes, dNTPs, and a buffer comprising buffering agents, salts, and crowding reagents. In other embodiments, the method comprises introducing to a sample 2) a solution comprising enzymes, dATP, dNTPs. and a buffering agent comprising salts, crowding reagents, and reducing reagents. In some embodiments, the method comprises incubating the sample with the primers and solution at a reaction temperature for a length of time. In some embodiments, the method produces single-stranded oligonucleotide amplicons according to a sequence comprising at least a portion of a complementary sequence of the FP and a sequence extending from and including at least a portion of the RP2.

In some embodiments, the enzymes comprise a recombinase enzyme, a single strand binding protein, and a strand displacing polymerase. In some embodiments, the recombinase enzyme is an() RecA. In other embodiments, the recombinase enzyme is a homologous protein of RecA including but not limited to Rad51 in eukaryotes, and RadA in archaea. In accordance with the methods described herein other recombinase enzymes may be used. e.g., recombinase enzymes in the RecA/Rad51 family of enzymes. In some embodiments, the single-stranded binding protein issingle-stranded DNA binding protein (EcSSB). In other embodiments, the single-stranded binding protein is a single-stranded binding protein from a virus; non-limiting examples include but are not limited to a GP32 protein from T4 phage, or an ICP8 protein from HSV-1. In some embodiments, the single-stranded binding protein is eukaryotic mitochondrial single-stranded binding protein (mtSSB). In accordance with the methods described herein other single-stranded binding proteins may be used. In some embodiments, the strand displacing polymerase isDNA polymerase I (Bsu). In other embodiments, the strand displacing polymerase isDNA Polymerase I (Bst). In further embodiments, the strand displacing polymerase is a Klenow Fragment. Non-limiting examples of strand displacing polymerases include but are not limited to phi29, T4 DNA polymerase and other mesophilic and psychrophilic DNA/RNA polymerases.

In some embodiments, the buffer may comprise buffering agents. As used herein a “buffering agent” refers to a weak acid or base solution used to maintain the acidity (pH) of a solution near a chosen value. Non-limiting examples of buffering agents that may be used in the buffer may include but are not limited to Tris, Tris-HCl, Tris Acetate. PBS, or a combination thereof.

In some embodiments, the buffer may comprise salts. Non-limiting examples of salt that may be used in the buffer may include but are not limited to sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl), sodium acetate (NaCHCOO), magnesium acetate (Mg(CHHO), monosodium phosphate (NaH2PO4), disodium phosphate (NAPO), or a combination thereof.

In some embodiments, the buffer may comprise a reducing agent. As used herein, a “reducing agent” refers to a substance that tends to bring about a reduction reaction by being oxidized and losing electrons. Non-limiting examples of reducing agents that may be used in the buffer may include but are not limited to dithiothreitol (OTT), tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof.

In some embodiments, the buffer may comprise a crowding agent. As used herein, a “crowding agent” refers to inert, non-charged polymers of certain sizes, that occupy space but do not interact with target proteins. Non-limiting examples of crowding agents that may be used in the buffer may include but are not limited to polyvinylpyrrolidone (PVP), or polyethylene glycol (PEG), Ficoll. Dextran, or a combination thereof.